Tocilizumab in Patients Hospitalized with Covid-19 Pneumonia.

BACKGROUND: Coronavirus disease 2019 (Covid-19) pneumonia is often associated with hyperinflammation. Despite the disproportionate incidence of Covid-19 among underserved and racial and ethnic minority populations, the safety and efficacy of the anti-interleukin-6 receptor antibody tocilizumab in patients from these populations who are hospitalized with Covid-19 pneumonia are unclear. METHODS: We randomly assigned (in a 2:1 ratio) patients hospitalized with Covid-19 pneumonia who were not receiving mechanical ventilation to receive standard care plus one or two doses of either tocilizumab (8 mg per kilogram of body weight intravenously) or placebo. Site selection was focused on the inclusion of sites enrolling high-risk and minority populations. The primary outcome was mechanical ventilation or death by day 28. RESULTS: A total of 389 patients underwent randomization, and the modified intention-to-treat population included 249 patients in the tocilizumab group and 128 patients in the placebo group; 56.0% were Hispanic or Latino, 14.9% were Black, 12.7% were American Indian or Alaska Native, 12.7% were non-Hispanic White, and 3.7% were of other or unknown race or ethnic group. The cumulative percentage of patients who had received mechanical ventilation or who had died by day 28 was 12.0% (95% confidence interval [CI], 8.5 to 16.9) in the tocilizumab group and 19.3% (95% CI, 13.3 to 27.4) in the placebo group (hazard ratio for mechanical ventilation or death, 0.56; 95% CI, 0.33 to 0.97; P = 0.04 by the log-rank test). Clinical failure as assessed in a time-to-event analysis favored tocilizumab over placebo (hazard ratio, 0.55; 95% CI, 0.33 to 0.93). Death from any cause by day 28 occurred in 10.4% of the patients in the tocilizumab group and 8.6% of those in the placebo group (weighted difference, 2.0 percentage points; 95% CI, -5.2 to 7.8). In the safety population, serious adverse events occurred in 38 of 250 patients (15.2%) in the tocilizumab group and 25 of 127 patients (19.7%) in the placebo group. CONCLUSIONS: In hospitalized patients with Covid-19 pneumonia who were not receiving mechanical ventilation, tocilizumab reduced the likelihood of progression to the composite outcome of mechanical ventilation or death, but it did not improve survival. No new safety signals were identified. (Funded by Genentech; EMPACTA ClinicalTrials.gov number, NCT04372186.).

The Association of Baseline Plasma SARS-CoV-2 Nucleocapsid Antigen Level and Outcomes in Patients Hospitalized With COVID-19.

BACKGROUND: Levels of plasma SARS-CoV-2 nucleocapsid (N) antigen may be an important biomarker in patients with COVID-19 and enhance our understanding of the pathogenesis of COVID-19. OBJECTIVE: To evaluate whether levels of plasma antigen can predict short-term clinical outcomes and identify clinical and viral factors associated with plasma antigen levels in hospitalized patients with SARS-CoV-2. DESIGN: Cross-sectional study of baseline plasma antigen level from 2540 participants enrolled in the TICO (Therapeutics for Inpatients With COVID-19) platform trial from August 2020 to November 2021, with additional data on day 5 outcome and time to discharge. SETTING: 114 centers in 10 countries. PARTICIPANTS: Adults hospitalized for acute SARS-CoV-2 infection with 12 days or less of symptoms. MEASUREMENTS: Baseline plasma viral N antigen level was measured at a central laboratory. Delta variant status was determined from baseline nasal swabs using reverse transcriptase polymerase chain reaction. Associations between baseline patient characteristics and viral factors and baseline plasma antigen levels were assessed using both unadjusted and multivariable modeling. Association between elevated baseline antigen level of 1000 ng/L or greater and outcomes, including worsening of ordinal pulmonary scale at day 5 and time to hospital discharge, were evaluated using logistic regression and Fine-Gray regression models, respectively. RESULTS: Plasma antigen was below the level of quantification in 5% of participants at enrollment, and 1000 ng/L or greater in 57%. Baseline pulmonary severity of illness was strongly associated with plasma antigen level, with mean plasma antigen level 3.10-fold higher among those requiring noninvasive ventilation or high-flow nasal cannula compared with room air (95% CI, 2.22 to 4.34). Plasma antigen level was higher in those who lacked antispike antibodies (6.42 fold; CI, 5.37 to 7.66) and in those with the Delta variant (1.73 fold; CI, 1.41 to 2.13). Additional factors associated with higher baseline antigen level included male sex, shorter time since hospital admission, decreased days of remdesivir, and renal impairment. In contrast, race, ethnicity, body mass index, and immunocompromising conditions were not associated with plasma antigen levels. Plasma antigen level of 1000 ng/L or greater was associated with a markedly higher odds of worsened pulmonary status at day 5 (odds ratio, 5.06 [CI, 3.41 to 7.50]) and longer time to hospital discharge (median, 7 vs. 4 days; subhazard ratio, 0.51 [CI, 0.45 to 0.57]), with subhazard ratios similar across all levels of baseline pulmonary severity. LIMITATIONS: Plasma samples were drawn at enrollment, not hospital presentation. No point-of-care test to measure plasma antigen is currently available. CONCLUSION: Elevated plasma antigen is highly associated with both severity of pulmonary illness and clinically important patient outcomes. Multiple clinical and viral factors are associated with plasma antigen level at presentation. These data support a potential role of ongoing viral replication in the pathogenesis of SARS-CoV-2 in hospitalized patients. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

Emerging Mechanisms of Pulmonary Vasoconstriction in SARS-CoV-2-Induced Acute Respiratory Distress Syndrome (ARDS) and Potential Therapeutic Targets.

The 1918 influenza killed approximately 50 million people in a few short years, and now, the world is facing another pandemic. In December 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an international outbreak of a respiratory illness termed coronavirus disease 2019 (COVID-19) and rapidly spread to cause the worst pandemic since 1918. Recent clinical reports highlight an atypical presentation of acute respiratory distress syndrome (ARDS) in COVID-19 patients characterized by severe hypoxemia, an imbalance of the renin-angiotensin system, an increase in thrombogenic processes, and a cytokine release storm. These processes not only exacerbate lung injury but can also promote pulmonary vascular remodeling and vasoconstriction, which are hallmarks of pulmonary hypertension (PH). PH is a complication of ARDS that has received little attention; thus, we hypothesize that PH in COVID-19-induced ARDS represents an important target for disease amelioration. The mechanisms that can promote PH following SARS-CoV-2 infection are described. In this review article, we outline emerging mechanisms of pulmonary vascular dysfunction and outline potential treatment options that have been clinically tested.

Alterations in cardiovascular function in an experimental model of lung fibrosis and pulmonary hypertension.

NEW FINDINGS: What is the central question of this study? We have evaluated changes in cardiovascular physiology using echocardiography in an experimental model of lung fibrosis. What is the main finding and its importance? Remarkably, we report changes in cardiovascular function as early as day 7, concomitant with evidence of vascular remodelling. We also report that isolated pulmonary arteries were hypercontractile in response to a thromboxane A2 agonist. These findings are significant because the development of pulmonary hypertension is one of the most significant predictors of mortality in patients with lung fibrosis, where there are no available therapies and a lack of animal models. ABSTRACT: Group III pulmonary hypertension is observed in patients with chronic lung diseases such as chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. Pulmonary hypertension (PH) develops as a result of extensive pulmonary vascular remodelling and resultant changes in vascular tone that can lead to right ventricle hypertrophy. This eventually leads to right heart failure, which is the leading indicator of mortality in patients with idiopathic pulmonary fibrosis. Treatments for group III PH are not available, in part owing to a lack of viable animal models. Here, we have evaluated the cardiovascular changes in a model of lung fibrosis and PH. Data obtained from this study indicated that structural alterations in the right heart, such as right ventricular wall hypertrophy, occurred as early as day 14, and similar increases in right ventricle chamber size were seen between days 21 and 28. These structural changes were correlated with decreases in the systolic function of the right ventricle and right ventricular cardiac output, which also occurred between the same time points. Characterization of pulmonary artery dynamics also highlighted that PH might be occurring as early as day 21, indicated by reductions in the velocity-time integral; however, evidence for PH is apparent as early as day 7, indicated by the significant reduction in pulmonary acceleration time values. These changes are consistent with evidence of vascular remodelling observed histologically starting on day 7. In addition, we report hyperactivity of bleomycin-exposed pulmonary arteries to a thromboxane A2 receptor (Tbxa2r) agonist.

Admission for COPD Exacerbation Is Associated with the Clinical Diagnosis of Pulmonary Hypertension: Results from a Retrospective Longitudinal Study of a Veteran Population.

Patients with chronic obstructive pulmonary disease and pulmonary hypertension (PH-COPD) have an increased risk of hospitalizations and death compared to COPD alone. Identifying PH in COPD is challenging because performing right heart catheterization, the gold standard for PH diagnosis, is invasive and not routinely performed. Clinical characterization of COPD patients at risk who are progressing toward PH will aid therapeutic development at earlier stages of progressively fatal PH-COPD. We studied the records of 5,45,086 patients in a large Veterans Affairs healthcare network (2000-2012) with a primary discharge diagnosis of COPD based on encounters' ICD-9 codes and further stratified into those who received an additional ICD-9 code for a PH diagnosis. Patients with PH-COPD were assigned to one of the four subgroups: those with (a) no history of exacerbation or hospital admissions, (b) history of exacerbations but no hospital admissions, (c) hospital admissions unrelated to COPD and (d) history of COPD exacerbation-related hospital admissions. We also examined the COPD and COPD-PH cohorts for associated comorbidities such as cardiac disease and the presence of obstructive sleep apnea (OSA). A regression analysis revealed that patients with COPD exacerbation-related hospital admissions had 7 × higher risk of having a concomitant clinical diagnosis of PH compared to non-hospitalized patients. COPD-PH patients had higher rates of cardiac comorbidities (89% vs. 66%) and OSA (34% vs. 16%) compared to COPD alone. We conclude that COPD patients hospitalized for COPD exacerbations are at a higher risk for developing PH, and hospitalized COPD patients with cardiac comorbidities and/or OSA should be screened as at-risk population for developing PH.

Altered Hypoxic-Adenosine Axis and Metabolism in Group III Pulmonary Hypertension.

Group III pulmonary hypertension (PH) is a highly prevalent and deadly lung disorder with limited treatment options other than transplantation. Group III PH affects patients with ongoing chronic lung injury, such as idiopathic pulmonary fibrosis (IPF). Between 30 and 40% of patients with IPF are diagnosed with PH. The diagnosis of PH has devastating consequences to these patients, leading to increased morbidity and mortality, yet the molecular mechanisms involved in the development of PH in patients with chronic lung disease remain elusive. Our hypothesis was that the hypoxic-adenosinergic system is enhanced in patients with group III PH compared with patients with IPF with no PH. Explanted lung tissue was analyzed for markers of the hypoxic-adenosine axis, including expression levels of hypoxia-inducible factor (HIF)-1A, adenosine A2B receptor, CD73, and equilibrative nucleotide transporter-1. In addition, we assessed whether altered mitochondrial metabolism was present in these samples. Increased expression of HIF-1A was observed in tissues from patients with group III PH. These changes were consistent with increased evidence of adenosine accumulation in group III PH. A novel observation of our study was of evidence suggesting altered mitochondrial metabolism in lung tissue from group III PH leading to increased succinate levels that are able to further stabilize HIF-1A. Our data demonstrate that the hypoxic-adenosine axis is up-regulated in group III PH and that subsequent succinate accumulation may play a part in the development of group III PH.

Clinical and Immunological Factors in Emphysema Progression. Five-Year Prospective Longitudinal Exacerbation Study of Chronic Obstructive Pulmonary Disease (LES-COPD).

RATIONALE: Cross-sectional studies of T-cell responses to self-antigens correlate with baseline emphysema severity. OBJECTIVES: We investigated whether clinical and/or immunological factors could predict disease progression, such as emphysema, FEV1, and 6-minute-walk distance (6MWD), in former and active smokers in a 5-year prospective study. METHODS: We recruited 224 ever smokers over 40 years of age and with greater than a 15 pack-year smoking history. MEASUREMENTS AND MAIN RESULTS: Repeated spirometry, 6MWD, and peripheral blood T-cell cytokine responses to lung elastin fragments were measured. Baseline and repeat chest computed tomography (CT) scans (34 to 65 mo apart) were used to quantify emphysema progression. Of the 141 ever-smokers with baseline and repeat CT scans, the mean (SD) annual rate of change in percent emphysema was +0.46 (0.92), ranging from -1.8 to +4.1. In multivariable analyses, the rate of emphysema progression was greater in subjects who had lower body mass index (BMI) (+0.15 per 5-unit decrease in BMI; 95% confidence interval, +0.03 to +0.29). In active smokers, increased IFN-γ and IL-6 T-cell responses had a positive association with the annual rate of emphysema progression. Male sex and IL-6 T-cell responses to elastin fragments were significantly associated with annual 6MWD decline, whereas IL-13 was associated with an increase in annual 6MWD. CONCLUSIONS: The rate of emphysema progression quantified by CT scans among ever-smokers was highly variable; clinical factors and biomarkers explained only some of the variability. Aggressive clinical care that targets active smokers with autoreactive T cells and low BMI may temporize progression of emphysema.

Regulation of IL-4 receptor signaling by STUB1 in lung inflammation.

RATIONALE: IL-4Rα, the common receptor component for IL-4 and IL-13, plays a critical role in IL-4- and IL-13-mediated signaling pathways that regulate airway inflammation and remodeling. However, the regulatory mechanisms underlying IL-4Rα turnover and its signal termination remain elusive. OBJECTIVES: To evaluate the role of STUB1 (STIP1 homology and U-Box containing protein 1) in regulating IL-4R signaling in airway inflammation. METHODS: The roles of STUB1 in IL-4Rα degradation and its signaling were investigated by immunoblot, immunoprecipitation, and flow cytometry. The involvement of STUB1 in airway inflammation was determined in vivo by measuring lung inflammatory cells infiltration, mucus production, serum lgE levels, and alveolar macrophage M2 activation in STUB1(-/-) mice. STUB1 expression was evaluated in airway epithelium of patients with asthma and lung tissues of subjects with chronic obstructive pulmonary disease. MEASUREMENTS AND MAIN RESULTS: STUB1 interacted with IL-4Rα and targeted it for ubiquitination-mediated proteasomal degradation, terminating IL-4 or IL-13 signaling. STUB1 knockout cells showed increased levels of IL-4Rα and sustained STAT6 activation, whereas STUB1 overexpression reduced IL-4Rα levels. Mice deficient in STUB1 had spontaneous airway inflammation, alternative M2 activation of alveolar macrophage, and increased serum IgE. STUB1 levels were increased in airways of subjects with asthma or chronic obstructive pulmonary disease, suggesting that up-regulation of STUB1 might be an important feedback mechanism to dampen IL-4R signaling in airway inflammation. CONCLUSIONS: Our study identified a previously uncharacterized role for STUB1 in regulating IL-4R signaling, which might provide a new strategy for attenuating airway inflammation.

Endothelium-dependent relaxations in the aorta from K(2p)6.1 knockout mice.

K2P6.1 or TWIK-2, a two-pore domain K channel, is an important regulator of cardiovascular function. K2P6.1 is highly expressed in vascular smooth muscle and endothelium. Mice (8-12 wk) lacking functional K2P6.1 (K2P6.1(-/-)) are hypertensive and have enhanced vascular contractility. It is not known whether the lack of functional K2P6.1 in endothelium has a role in the vascular dysfunction in K2P6.1(-/-) mice. We tested the hypothesis: K2P6.1(-/-) mice have impaired endothelium-dependent relaxations. K2P6.1(-/-) mice were ∼35 mmHg more hypertensive than WT mice at both 8-12 wk (young adult) and 20-24 wk (mature mice, P < 0.01; n = 8-10). Endothelium-dependent relaxations of the thoracic aorta were evaluated by isometric myography after contraction with phenylephrine (10(-6) M). Maximal ACh-dependent relaxations were increased from 65 ± 1% to 73 ± 1% in the aorta from young adult (P < 0.01; n = 6) and from 45 ± 1% to 74 ± 1% in the aorta from mature (P < 0.001; n = 5) K2P6.1(-/-) mice compared with K2P6.1(+/+) littermates. However, in the aorta from young adult and mature K2P6.1(+/+) mice, 10(-5) M indomethacin, a cyclooxygenase inhibitor, increased maximal ACh relaxations to knockout levels. Enhanced relaxation was also seen with ATP, a P2Y purinergic agonist, and A23187, a nonreceptor-based agonist in mature K2P6.1(-/-) mice. Mature adult aorta from K2P6.1(-/-) showed an attenuated ACh-mediated contraction in the presence of nitro-l-arginine methyl ester (l-NAME) and without precontraction of 0.97 mN vs. 7.5 mN in K2P6.1(-/-) and K2P6.1(+/+) (P < 0.001; n = 5). In summary, K2P6.1(-/-) mice, which are hypertensive, have enhanced endothelium-dependent relaxations in the aorta due to the suppression of an indomethacin-sensitive constrictor component.

Association of Glucose-6-Phosphate Dehydrogenase Deficiency With Outcomes in US Veterans With COVID-19.

Importance: The underlying biological risk factors for severe outcome due to SAR-CoV-2 infection are not well defined. Objective: To determine the association between glucose-6-phosphate dehydrogenase (G6PD) deficiency and severity of COVID-19. Design, Setting, and Participants: This retrospective cohort study included analysis of 24 700 veterans with G6PD enzyme testing prior to January 1, 2020, obtained through the US Veterans Health Administration national databases. These veterans were cross-referenced with the Veterans Administration COVID-19 Shared Data Resource for SARS-CoV-2 testing from February 15, 2020, to January 1, 2021. The final study population consisted of 4811 veterans who tested positive for SARS-CoV-2. Statistical analysis was performed from June to December 2021. Exposures: G6PD deficiency. Main Outcomes and Measures: COVID-19 severe illness, as defined by the Centers for Disease Control and Prevention: hospitalization, need for mechanical ventilation and/or intensive care unit admission, or in-hospital mortality after a positive SARS-CoV-2 test. Results: Among 4811 veterans in the Veterans Health Administration who had historical G6PD enzyme activity test results and SARS-CoV-2 positivity included in this study, 3868 (80.4%) were male, 1553 (32.3%) were Black, and 1855 (39%) were White; 1228 (25.5%) were 65 years or older and 3583 (74.5%) were younger than 65 years. There were no significant differences in age, body mass index, or Charlson Comorbidity Index were present between the veterans with G6PD deficiency and without G6PD deficiency. Among these veterans with SARS-CoV-2 infection, G6PD deficiency was more prevalent in Black male veterans (309 of 454 [68.1%]) compared with other racial and ethnic groups. Black male veterans less than 65 years of age with G6PD deficiency had approximately 1.5-fold increased likelihood of developing severe outcomes from SARS-CoV-2 infection compared with Black male veterans without G6PD deficiency (OR, 1.47; 95% CI, 1.03-2.09). In the small subset of White male veterans with G6PD deficiency, we observed an approximately 3.6-fold increased likelihood of developing severe outcomes from SARS-CoV-2 infection compared with White male veterans aged 65 years or older without G6PD deficiency (OR, 3.58; 95% CI, 1.64-7.80). This difference between veterans with and without G6PD deficiency was not observed in younger White male veterans or older Black male veterans, nor in smaller subsets of other male veterans or in female veterans of any age. Conclusions and Relevance: In this cohort study of COVID-19-positive veterans, Black male veterans less than 65 years of age and White male veterans 65 years of age or older with G6PD deficiency had an increased likelihood of developing severe COVID-19 compared with veterans without G6PD deficiency. These data indicate a need to consider the potential for G6PD deficiency prior to treatment of patients with SARS-CoV-2 infection as part of clinical strategies to mitigate severe outcomes.

Treatment of Severe COVID-19 Infection With Remdesivir in Peritoneal Dialysis.

End-stage kidney disease (ESKD) has been shown to be correlated with an increased risk of COVID-19 infection and mortality. Remdesivir is an effective non-EUA U.S. Food and Drug Administration (FDA)-approved antiviral agent for the treatment of COVID-19 in hospitalized adult and pediatric patients, though a lack of data has prevented its use in patients with severe kidney disease including dialysis patients. Some observational studies report the use of remdesivir in hemodialysis patients, but there are no reports of patients treated with remdesivir on peritoneal dialysis. Dialysis modalities may affect drug pharmacokinetics, and safety and efficiency of remdesivir in peritoneal dialysis is unknown. We report the first case, to our knowledge, of using remdesivir in a patient treated with peritoneal dialysis with no significant adverse events. This case illustrates the potential for remdesivir to be considered in peritoneal dialysis patients with severe COVID infection. Proper risk analysis and careful monitoring should be done, given the unpredictable clearance of the drug.

The physiologic aggresome mediates cellular inactivation of iNOS.

Nitric Oxide (NO), produced by inducible nitric oxide synthase (iNOS), has been implicated in the pathogenesis of various biological and inflammatory disorders. Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed "physiologic aggresome," is exemplified by aggresome formation of iNOS, an important host defense protein. The functional significance of cellular formation of the iNOS aggresome is hitherto unknown. In this study, we used live cell imaging, fluorescence microscopy, and intracellular fluorescence NO probes to map the subcellular location of iNOS and NO under various conditions. We found that NO production colocalized with cytosolic iNOS but aggresomes containing iNOS were distinctly devoid of NO production. Further, cells expressing iNOS aggresomes produced significantly less NO as compared with cells not expressing aggresomes. Importantly, primary normal human bronchial epithelial cells, stimulated by cytokines to express iNOS, progressively sequestered iNOS to the aggresome, a process that correlated with marked reduction of NO production. These results suggest that bronchial epithelial cells used the physiologic aggresome mechanism for iNOS inactivation. Our studies reveal a novel cellular strategy to terminate NO production via formation of the iNOS aggresome.

Implementation of a Virtual Huddle to Support Patient Care During the COVID-19 Pandemic.

Background: During a surge of COVID-19 cases, the volume of acute care patients with hypoxemic respiratory failure placed a high burden of responsibility on internal medicine, pulmonary and critical care medicine, and clinical pharmacy services. Observations: We describe the COVID-19 Tele-Huddle Program, a novel approach to communication between key stakeholders in COVID-19 patient care through a daily video conferencing huddle. The program was implemented during a 4-week surge in COVID-19 cases at a large, academic medical center in Houston, Texas. Data collected during the COVID-19 Tele-Huddle Program included the type and number of interventions implemented, number of patients discussed, and COVID-19 therapies provided. In addition, hospital medicine team members completed a user-experience survey. Conclusions: A multidisciplinary consultation service using video conferencing can support the care of patients with high disease severity without overwhelming existing inpatient medical, intensive care, and pharmacy services.

Src kinase-mediated phosphorylation stabilizes inducible nitric-oxide synthase in normal cells and cancer cells.

Src kinases are key regulators of cellular proliferation, survival, motility, and invasiveness. They play important roles in the regulation of inflammation and cancer. Overexpression or hyperactivity of c-Src has been implicated in the development of various types of cancer, including lung cancer. Src inhibition is currently being investigated as a potential therapy for non-small cell lung cancer in Phase I and II clinical trials. The mechanisms of Src implication in cancer and inflammation are linked to the ability of activated Src to phosphorylate multiple downstream targets that mediate its cellular effector functions. In this study, we reveal that inducible nitric-oxide synthase (iNOS), an enzyme also implicated in cancer and inflammation, is a downstream mediator of activated Src. We elucidate the molecular mechanisms of the association between Src and iNOS in models of inflammation induced by lipopolysaccharide and/or cytokines and in cancer cells and tissues. We identify human iNOS residue Tyr(1055) as a target for Src-mediated phosphorylation. These results are shown in normal cells and cancer cells as well as in vivo in mice. Importantly, such posttranslational modification serves to stabilize iNOS half-life. The data also demonstrate interactions and co-localization of iNOS and activated Src under inflammatory conditions and in cancer cells. This study demonstrates that phosphorylation of iNOS by Src plays an important role in the regulation of iNOS and nitric oxide production and hence could account for some Src-related roles in inflammation and cancer.

Idiopathic pulmonary fibrosis and pulmonary hypertension: Heracles meets the Hydra.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease where the additional presence of pulmonary hypertension (PH) reduces survival. In particular, the presence of coexistent pulmonary vascular disease in patients with advanced lung parenchymal disease results in worse outcomes than either diagnosis alone. This is true with respect to the natural histories of these diseases, outcomes with medical therapies, and even outcomes following lung transplantation. Consequently, there is a striking need for improved treatments for PH in the setting of IPF. In this review, we summarize existing therapies from the perspective of molecular mechanisms underlying lung fibrosis and vasoconstriction/vascular remodelling and discuss potential future targets for pharmacotherapy. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.

Extracellular Vesicles Attenuate Nitrofen-Mediated Human Pulmonary Artery Endothelial Dysfunction: Implications for Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) leads to pathophysiologic pulmonary vasoreactivity. Previous studies show that mesenchymal stromal cell-derived extracellular vesicles (MSCEv) inhibit lung inflammation and vascular remodeling. We characterize MSCEv and human pulmonary artery endothelial cell (HPAEC) interaction, as well as the pulmonary artery (PA) response to MSCEv treatment. HPAECs were cultured with and without exposure to nitrofen (2,4-dichloro-phenyl-p-nitrophenylether) and treated with MSCEv. HPAEC viability, architecture, production of reactive oxygen species (ROS), endothelial dysfunction-associated protein levels (PPARγ, LOX-1, LOX-2, nuclear factor-κB [NF-κB], endothelial NO synthase [eNOS], ET-1 [endothelin 1]), and the nature of MSCEv-cellular interaction were assessed. Newborn rodents with and without CDH (nitrofen model and Sprague-Dawley) were treated with intravascular MSCEv or vehicle control, and their PAs were isolated. Contractility was assessed by wire myography. The contractile (KCL and ET-1) and relaxation (fasudil) responses were evaluated. HPAEC viability correlated inversely with nitrofen dose, while architectural compromise was directly proportional. There was a 2.1 × increase in ROS levels in nitrofen HPAECs (P < 0.001), and MSCEv treatment attenuated ROS levels by 1.5 × versus nitrofen HPAECs (P < 0.01). Nitrofen-induced alterations in endothelial dysfunction-associated proteins are shown, and exposure to MSCEv restored more physiologic expression. Nitrofen HPAEC displayed greater MSCEv uptake (80% increase, P < 0.05). Adenosine, a clathrin-mediated endocytosis inhibitor, decreased uptake by 46% (P < 0.05). CDH PA contraction was impaired with KCL (108.6% ± 1.4% vs. 112.0% ± 1.4%, P = 0.092) and ET-1 (121.7% ± 3.0% vs. 131.2% ± 1.8%, P < 0.01). CDH PA relaxation was impaired with fasudil (32.2% ± 1.9% vs. 42.1% ± 2.2%, P < 0.001). After MSCEv treatment, CDH PA contraction improved (125.9% ± 3.4% vs. 116.4 ± 3.5, P = 0.06), and relaxation was unchanged (32.5% ± 3.2% vs. 29.4% ± 3.1%, P = 0.496). HPAEC exposure to nitrofen led to changes consistent with vasculopathy in CDH, and MSCEv treatment led to a more physiologic cellular response. MSCEv were preferentially taken up by nitrofen-treated cells by clathrin-dependent endocytosis. In vivo, MSCEv exposure improved PA contractile response. These data reveal mechanisms of cellular and signaling alterations that characterize MSCEv-mediated attenuation of pulmonary vascular dysfunction in CDH-associated pulmonary hypertension.

Twik-2-/- mouse demonstrates pulmonary vascular heterogeneity in intracellular pathways for vasocontractility.

We have previously shown Twik-2-/- mice develop pulmonary hypertension and vascular remodeling. We hypothesized that distal pulmonary arteries (D-PAs) of the Twik-2-/- mice are hypercontractile under physiological venous conditions due to altered electrophysiologic properties between the conduit and resistance vessels in the pulmonary vascular bed. We measured resting membrane potential and intracellular calcium through Fura-2 in freshly digested pulmonary artery smooth muscles (PASMCs) from both the right main (RM-PA) and D-PA (distal) regions of pulmonary artery from WT and Twik-2-/- mice. Whole segments of RM-PAs and D-PAs from 20 to 24-week-old wildtype (WT) and Twik-2-/- mice were also pressurized between two glass micropipettes and bathed in buffer with either arterial or venous conditions. Abluminally-applied phenylephrine (PE) and U46619 were added to the buffer at log increments and vessel diameter was measured. All values were expressed as averages with ±SEM. Vasoconstrictor responses did not differ between WT and Twik-2-/- RM-PAs under arterial conditions. Under venous conditions, Twik-2-/- RM-PAs showed an increased sensitivity to PE with a lower EC50 (P = 0.02). Under venous conditions, Twik-2-/- D-PAs showed an increase maximal vasoconstrictor response to both phenylephrine and U46619 compared to the WT mice (P < 0.05). Isolated PASMCs from Twik-2 -/- D-PA were depolarized and had higher intracellular calcium levels compared to PASMCs from RM-PA of both WT and Twik-2-/- mice. These studies suggest that hypercontractile responses and electrophysiologic properties unique to the anatomic location of the D-PAs may contribute to pulmonary hypertensive vasculopathy.

Hemodynamic and Pathologic Characterization of the TASK-1-/- Mouse Does Not Demonstrate Pulmonary Hypertension.

INTRODUCTION: Pulmonary hypertension (PH) carries significant associated morbidity and mortality and the underlying molecular mechanisms of PH are not well understood. Loss-of-function mutations in TASK-1 potassium channels are associated with PH in humans. Although TASK-1 has been considered in the development of PH for over a decade, characterization of TASK-1 knockout mice has been limited to in vitro studies or in vivo studies in room air at isolated time points. The purpose of this study was twofold. First, we sought to determine if TASK-/- male and female mice developed PH over the span of one year. Second, we sought to determine the effect of chronic hypoxia, a stimulus for PH, and its recovery on PH in TASK-1-/- mice. METHODS: We measured right ventricular systolic pressure (RVSP) and vascular remodeling in male and female C57BL/6 WT and TASK-1-/- mice at separate time points: 20-24 weeks and 1 year of age. Additionally, we measured RVSP and vascular remodeling in TASK-1-/- and wild-type mice between 13 and 16 weeks of age exposed to 10% hypoxia for 3 weeks followed by recovery to room air conditions for an additional 6 weeks. RESULTS: RVSP was similar between WT and TASK-/- mice. Male and female WT and TASK-1-/- mice all demonstrated age-related increases in RVSP, which correlated to age-related vascular remodeling in male mice but not in female mice. Male TASK-1-/- and WT mice exposed to chronic hypoxia demonstrated increased RVSP, which decreased following room air recovery. WT and TASK-1-/- male mice demonstrated vascular remodeling upon exposure to hypoxia that persisted in room air recovery. CONCLUSION: Female and male TASK-1-/- mice do not develop hemodynamic or vascular evidence for PH, but RVSP rises in an age-dependent manner independent of genotype. TASK-1-/- and WT male mice develop hypoxia-induced elevations in RVSP that decrease to baseline after recovery in room air. TASK-1-/- and WT male mice demonstrate vascular remodeling after exposure to hypoxia that persists despite recovery to room air conditions and does not correlate with RVSP normalization.

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice. Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software. Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.